Synthesis of highly potent lymphocyte function-associated antigen-1 antagonists labeled with carbon-14 and with stable isotopes, part 3

Article abstract of DOI:10.1002/jlcr.3698

The drug candidates (2) and (3) are highly potent LFA-1 inhibitors. They were efficiently prepared labeled with carbon-14 using a palladium-catalyzed carboxylation of an iodo-precursor (5) and sodium formate-¹⁴C to afford acid [¹⁴C]-

Full text of DOI:10.1002/jlcr.3698

Synthesis of highly potent Lymphocyte Function-Associated Antigen-1 Antagonists
Labeled with Carbon-14 and with Stable Isotopes, Part 3
Bachir Latli*, Matt Hrapchak, Guisheng Li, Jon Lorenz, Josh Horan, Carl A. Busacca, and
Chris H. Senanayake
Chemical Development, Boehringer Ingelheim Pharmaceuticals, Inc.
900 Ridgebury Road, Ridgefield, Connecticut 06877 USA.
Abstract:
The drug candidates (2) and (3) are highly potent LFA-1 inhibitors. They were efficiently
prepared labeled with carbon-14 using a palladium-catalyzed carboxylation of an iodo-
precursor (5) and sodium formate-14C to afford acid [14C]-(6), which was coupled via an
amide bond to chiral amines (7) and (8) in 52% and 48% overall yield respectively and with
specific activities higher than 56 mCi/mmol and radiochemical purities of 99%. For stable
isotopes synthesis, the amine [2H8]-(7) was synthesized in three steps from 2-cyanopyridine-
2H4 using Kulinkovich-Szymonik aminocyclopropanation, followed by coupling to L-
alanine-2,3,3,3-2H4-N-t-BOC, and then removal of the BOC-protecting group. Amide bond
formation with acid (6) gave [2H8]-(2) in 36% overall yield. The amine [13C4,15N]-(8) was
obtained in two steps using L-threonine-14C4,15N and then coupled to acid [13C]-(6) to give
[13C5,15N]-(3) in 56% overall yield.
Keywords: LFA-1, carbon-14, carbon-13, nitrogen-15, deuterium, radiosynthesis
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3698
This article is protected by copyright. All rights reserved.

Introduction:
The lymphocyte function associated antigen-1 (LFA-1) is an essential component in normal
immune system function and a target for drug discovery due to its broad therapeutic potential
in treating inflammatory diseases. We have previously described the synthesis of some
potent LFA-1 antagonists, including compound (1) (Figure 1).1 The continuous search for
compounds with higher potency and better pharmacokinetics led to the discovery of
compounds (2) and (3), in which the sulfonamide was replaced with an amide linkage, a
fluorine atom was introduced on the dichlorophenyl moiety to suppress oxidative metabolism,
and the trifluoromethoxy group was replaced with a nitrile group.2 Further modifications on
compound (2) included replacing the methyl group with a chiral 2-propanol moiety, and the
pyridine ring with (1,3-dimethylpyrazole-4-yl)pyrimidine in compound (3),2 Figure 1.
Herein, we report the synthesis of compounds (2) and (3) labeled with carbon-14 and with
stable isotopes.
Results and discussion:
Carbon-14 labeled (2) and (3), Scheme 1, were prepared in two radioactive steps in 52% and
48% radiochemical overall yield respectively. The chiral bicyclic imidazole (4) accessible
from 3,5-dichloro-4-fluoroaniline according to published procedures,3 was iodinated using
one equivalent of N-iodosuccinimide and pyridinium p-toluenesulfonate (PPTS, 0.1
equivalent) in methylene chloride. The iodination gave about 10% of a diiodo-byproduct
which was removed by silica gel chromatography using 100% toluene as the eluting solvent.
The direct and selective palladium-catalyzed carboxylation of iodide (5) using sodium
formate-14C gave carbon-14 labeled carboxylic acid derivative [14C]-(6) in 70% yield. This
selective
carboxylation,
which
presumably
involves
palladium
catalyzed
hydroxycarbonylation of in-situ generated carbon monoxide was suitable for this substrate
which contains two chlorine atoms and a nitrile group susceptible to strong acids and
bases.2,4-7 An outstanding review on recent developments in carbonylation chemistry should
be consulted as well.8 Finally, amide bond formation between the acid [14C]-(6) and the chiral
amines (7) and (8) gave the desired [14C]-(2) and [14C]-(3) respectively with specific
activities higher than 56 mCi/mmol and radiochemical purities of 99%. Several conditions for
the amide bond formation were tried.2 in our hands higher yields were obtained when using
acylchloride derivatives.
This article is protected by copyright. All rights reserved.

As shown in Figure 1, both compounds contain two chlorine atoms; it was necessary that the
stable isotope labeled compounds should be least 6 atomic mass units higher to easily
distinguish them from the unlabeled drug candidates by mass spectroscopy. To prepare
deuterium labeled (2), (S)-2-amino-N-(1-pyridin-2H4-2-yl-cyclopropyl)-propionamide [2H8]-
(7) was prepared in three steps and in 50% overall yield starting from the commercially
available 2-cyanopyridine-2H4, [2H4]-(9). The cyanopyridine was first converted to 1-pyridin-
2H4 cyclopropylamine [2H4]-(10) using ethyl magnesium bromide and titanium
tetraisopropoxide via Kulinkovich-Szymonik aminocyclopropanation.9,10 Coupling to BOC-
L-alanine-2H4, [2H4]-(11) using EDC and HOBT in THF gave [2H8]-(12). Finally, removal of
the BOC-protecting group and coupling to the acid (6) gave deuterium labeled (2) with very
high isotopic enrichment , see Figure 2, (Scheme 2).
The availability of L-threonine-13C4,15N commercially, allowed the preparation of the chiral
amine [13C4,15N]-(8) in two steps with 80% overall yield. L-Threonine-13C4,15N was first
BOC-protected using aqueous sodium bicarbonate and methanol in quantitative yield.11
Coupling to the amine (14)12 using EDC and HOBT in methylene chloride as seen before
afforded [13C4,15N]-(15) in good yield. Removal of the BOC-protecting group gave
[13C4,15N]-(8) in quantitative yield. The acid [13C]-(6), which was prepared during the cold
trials of carbon-14 synthesis was used in the final amide bond formation with the amine
[13C4,15N]-(8) to give [13C5,15N]-(3) in very good yield, (Scheme 3).
Experimental
Materials and methods
Liquid scintillation counting was accomplished using a Beckman LS6500TA and Ready
SafeTM cocktail (Beckman, Fullerton, CA, USA). NMR spectra were recorded with a Bruker
400 and 500 MHz spectrometers using either deuterated chloroform or deuterated DMSO as a
solvent and tetramethyl silane as the internal standard unless stated otherwise. Disposable
silica gel columns were obtained from TELEDYNE-ISCO (Lincoln, NE, USA). HPLC was
performed using Zorbax XDB C8 and 20% to 100% MeCN/water (both solvents contain 10
mM TFA) in 22 min runs, unless indicated otherwise. The chemicals 2-cyanopyridine-2H4
(99.6 atom % 2H) and L-alanine-2,3,3,3-2H4-N-t-BOC (99.2 atom % 2H) were purchased from
CDN (Pointe Claire Quebec, Canada). L-Threonine-13C4,15N (99 atom % 13C and 98.5
atom % 15N) and sodium formate-13C (99.5 atom % 13C) were obtained from ISOTEC
(Sigma-Aldrich, Miamisburg, OH, USA). Carbon-14 sodium formate was obtained from
This article is protected by copyright. All rights reserved.

American Radiolabeled Compounds (St. Louis, MO, USA) and from ViTrax (Placentia, CA,
USA). The rest of the reagents were purchased from Sigma-Aldrich Company (Milwaukee,
WI, USA).
Synthesis of [14C]-(2) and [14C]-(3):
(R)-4-((1-(3,5-dichloro-4-fluorophenyl)-5-iodo-3-methyl-2-oxo-2,3-dihydro-1H-
imidazo[1,2-a]imidazol-3-yl)methyl)benzonitrile, (5): To a solution of (4) (5.0 g, 12 mmol)
in anhydrous CH2Cl2 (100 mL), was added PPTS (0.31 g, 1.2 mmol) and the solution was
cooled in an ice-bath under nitrogen atmosphere. N-Iodosuccinimide (2.9 g, 12.2 mmol) was
o
added in four portions in a 30 minute period at 0 oC. The mixture was stirred at 0 C for 2 h
in the dark. LCMS showed 29% remaining of starting material. The ice bath was removed
and the dark mixture was further stirred for another 14 h at ambient temperature. A solution
of 5% aqueous Na2S2O3 (200 mL) was added and stirred for 15 minutes to give a light yellow
mixture, which was extracted with CH2Cl2 (100 mL ×2). The combined extracts were washed
with water (200 mL), dried over MgSO4, filtered and concentrated in vacuo to give 7.0 g of
yellow foam. Purification by flash chromatography using a silica gel column (10'' and 2.5''
diameter silica gel) packed in hexanes and eluting with 100% toluene gave few fractions of
the product contaminated with a diiodo-byproduct, which were combined and concentrated in
vacuo to give 0.72 g of material. The fractions containing the desired product were combined
o
and concentrated in vacuo at 45 C to give 4.8 g of an off-white solid in 74% yield. TLC
(100% toluene), Rf of the desired product = 0.13, Rf of the diiodo-byproduct = 0.2, the
starting material remains on the base line. LCMS: Rt = 2.03 min, MH+ = 541.4 (100%).
HPLC: Rt= 7.22 min, 99%. 1HNMR (400 MHz, CDCl3) 7.67(d, J = 5.8 Hz, 2H), 7.45(d, J
= 8.3 Hz, 2H), 7.05(d, J = 8.3 Hz, 2H), 6.97(s, 1H), 3.66(d, J = 13.84 Hz, 1H), 3.34(d, J =
13
13.84 Hz, 1H), 1.95(s, 3H). C NMR (101 MHz, CDCl3) : 173.94, 154.09, 151.6, 148.6,
138.6, 136.0, 132.4, 130.3, 123.0(m), 122.3, 118.1, 112.1, 75.7(m), 68.2, 56.7, 42.4, 22.4.
(R)-5-(4-Cyano-benzyl)-7-(3,5-dichloro-4-fluoro-phenyl)-5-methyl-6-oxo-6,7-dihydro-
5H-imidazo[1,2-a]imidazole-3-carboxylic-14C acid, [14C]-(6): Anhydrous DMF (2.0 mL)
was added to a thick walled screw cap tube containing carbon-14 sodium formate (200 mCi),
followed by Hünig's base (diisopropylethyl amine, DIPEA) (0.55 mL, 3.12 mmol) and acetic
anhydride (306 L, 3.18 mmol). After stirring for 30 min at room temperature, the mixture
became thick. In a separate flask, LiCl (208 mg, 4.90 mmol) was dissolved in anhydrous
DMF (4 mL) using sonication. To this, were added the iodide (5) (857 mg, 1.58 mmol) and
Pd(OAc)2 (74 mg, 0.33 mmol). The resulting solution was added to the tube containing
This article is protected by copyright. All rights reserved.

o
carbon-14 sodium formate mixture and the tube was heated to 80 C and stirred for 14 h. An
HPLC sample showed the acid [14C]-(6) as the only peak in the chromatogram and co-eluted
with the unlabeled (6) at 13.3 min. The dark mixture was transferred to a 100 mL Erlenmeyer
flask using EtOAc (40 mL) and treated with 2.75 N of aqueous HCl (30 mL) with stirring.
The organic phase was removed and the aqueous was extracted twice with EtAOc (40 mL
×2). The combined extracts were dried over Na2SO4, filtered and concentrated in vacuo to
give 1.3 g of viscous oil. Purification by chromatography using 40 g disposable silica gel
column and eluting with 10% to 20% EtOAc:CH2Cl2, then with 10% MeOH/CH2Cl2 gave
511 mg (70% based on the iodide) of the pure product. A total of 69 mCi was obtained.
HPLC: tR = 13.29 min. 96% chemical purity and 97% radiochemical purity.
(R)-3-(4-Cyanobenzyl)-1-(3,5-dichloro-4-fluorophenyl)-3-methyl-2-oxo-N-((S)-1-oxo-1-
((1-(pyridin-2-yl)cyclopropyl)amino)propan-2-yl)-2,3-dihydro-1H-imidazo[1,2-
a]imidazole-5-carboxamide-14C, [14C]-(2): To a solution of [14C]-(6) (511 mg, 1.1 mmol, 69
mCi) and (7) (106 mg, 1.1 mmol) in anhydrous DMF (10 mL) was added HATU (418 mg,
1.2 mmol) in one portion. To this solution, Hünig's base (1 mL, 5.5 mmol) was added
dropwise and the reaction was stirred at room temperature for 14 h. The reaction was
o
concentrated in vacuo at 35 C to remove most of the DMF, then diluted with CHCl3 (100
mL) and washed with water (100 mL). The organic phase was removed and dried over
Na2SO4, then filtered and concentrated in vacuo to give 0.85 g of foam. Purification by flash
chromatography using a 40 g disposable silica gel column and 0→10% MeOH/CH2Cl2 gave
531 mg of white foam in 74% yield (48 mCi). HPLC: [14C]-(2) eluted at 13.5 min and co-
eluted with unlabeled (2) with 99.56 % chemical purity and 99.93% radiochemical purity.
1
Specific activity of 58.6 mCi/mmol. HNMR (500 MHz, CDCl3) : 8.88 (exch, 1H), 8.47(s,
1H), 8.24 (exch, 1H), 7.64(d, J = 5.4 Hz, 2H), 7.57(m, 1H), 7.41(d, J = 8.3 Hz, 2H), 7.31(m,
2H), 7.14(d, 1H), 7.08(m, 1H), 7.01(d, J = 8.2 Hz, 2H), 6.94(s, 1H), 4.46(m, 1H), 4.14(d, J =
13.6 Hz, 1H), 3.28(d, J = 13.6 Hz, 1H), 1.93(s, 3H), 1.65(m, 2H), 1.59(d, J = 7.2 Hz, 3H),
1.26(m, 2H).
(R)-3-(4-Cyanobenzyl)-1-(3,5-dichloro-4-fluorophenyl)-N-((2S,3R)-1-((1-(5-(1,3-
dimethyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclopropyl)amino)-3-hydroxy-1-oxobutan-2-
yl)-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[1,2-a]imidazole-5-carboxamide-14C, [14C]-
(3): [14C]-(6) (458 mg, 0.99 mmol, 56 mCi) was dissolved in anhydrous THF (10 mL). Two
drops of DMF were added and the solution was cooled in an ice-bath. Oxalyl chloride (152
mg, 1.2 mmol) was added dropwise and the resulting was warmed gradually to room
This article is protected by copyright. All rights reserved.

temperature and stirred for 4 h. A sample was taken and quenched with morpholine. HPLC
indicated only a morpholine derivative was observed. The reaction was concentrated in vacuo
to foam. The amine (8) (367 mg, 1.0 mmol) was suspended in anhydrous THF (8 mL).
Hünig's base (0.34 mL, 2 mmol) was added dropwise at room temperature to give a colorless
solution with some white precipitate at the bottom of the flask. The solution was cooled in an
ice bath. The acylchloride derivative of [14C]-(6) was dissolved in THF (5 mL) and then
o
added dropwise to the flask containing the amine (8). After stirring for 1 h at 0 C, the ice-
bath was removed and the mixture was stirred for 14 h. Water (40 mL) was added and the
organic was extracted with MTBE (50 mL ×2). The combined organic layers were washed
with 2% aqueous NaHCO3 (50 mL ×2), dried over Na2SO4, filtered and concentrated in
vacuo to give 0.91 g of yellow solid. HPLC showed about 87% pure product by UV (230 nm)
or 90% at 254 nm. Purification using a 40 g silica gel column and up to 10% MeOH/CH2Cl2
gave 532 mg of an off-white solid with a specific activity 56.02 mCi/mmol and total activity
of 38.53 mCi in 69 % yield. HPLC (Halo C18, 2.1 x 50 mm, 2.7 M), A: water (0.1% TFA),
B: MeCN, 20 to 100% B in 9 min, injection volume 2 L, UV 254 nm, Rt = 6.32 (99.5%) and
1
99.7% radiochemical purity. HNMR(500 MHz, DMSO-d6) : 8.85(exch, 1H), 8.71(s, 2H),
8.25(exch, 1H), 8.05(s, 1H), 7.85(s, 1H), 7.81(d, J = 5.1 Hz, 2H), 7.61(d, J = 8.1 Hz, 2H),
7.02(d, J = 8.1 Hz, 2H), 5.12(exch, 1H), 4.55(m, 1H), 4.20(m,1H), 4.0(d, J = 13.8 Hz, 1H),
3.78(s, 3H), 3.48(d, J = 13.8 Hz, 1H), 2.29(s, 3H), 1.91(s, 3H), 1.56(m, 2H), 1.30(d, J = 7 Hz,
3H), 1.25(m, 2H).
Synthesis of [2H8]-(2):
1-Pyridin-2H4-2-yl-cyclopropylamine, [2H4]-(10): To a 100 mL round bottom flask
containing bis[2-(N,N-dimethylamino)ethyl]ether (4.0 mL, 20.36 mmol) and anhydrous THF
o
(6 mL) at -15 C, was added a solution of EtMgBr (21 mL, 1 M in THF) over a 90 minute-
period. After the addition was completed, the white mixture was warmed to 0 oC for 15 min.
In a separate 250 mL round bottom flask, a solution of 2-cyanopyridine-2H4 [2H4]-(9) (1 g,
9.25 mmol) in THF (20 mL) was heated to 50 oC in an oil bath. A solution of Ti(OiPr)4 (3.25
mL, 11.1 mmol) was added to this heated solution under a nitrogen atmosphere followed by
addition of the EtMgBr suspension via cannula. A 10 mL THF rinse was used to transfer the
remaining EtMgBr mixture to the reaction flask. The reaction turned yellow and quickly
darkened with reflux. The mixture thickened. After 2 h, HPLC showed most of the starting
material was consumed. The reaction was cooled to room temperature and treated with a
solution of 1N NaOH (30 mL) to give a yellow solution and a white precipitate. Me-THF (50
This article is protected by copyright. All rights reserved.

mL) was added and the organic phase was decanted, the solids were extracted with Me-THF
(30 mL ×2) and the combined extracts were concentrated in vacuo to about 30 mL and used
as it is in the next step. LCMS: one single peak, MH+ = 161.13.
[(S)-1-(1-Pyridin-2H4-2-yl-cyclopropylcarbamoyl)-ethyl-2H4]-carbamic acid tert-butyl
ester, [2H8]-(12): To a solution of crude [2H4]-(10) in Me-THF (30 mL) was added L-
alanine-2,3,3,3,-2H4-N-t-Boc (1.55 g, 8 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 1.1 g,
8.14 mmol), triethylamine (TEA, 1.12 mL, 8 mmol), and N-(3-diamethylaminopropyl)-N'-
ethylcarbodiimide hydrochloride (EDC, 1.55 g, 8.1 mmol). The reaction was stirred for 14 h
at room temperature. Water (50 mL) was added and the aqueous was extracted with CH2Cl2
(50 mL ×3). The combined extracts were washed with a saturated solution of NaHCO3 (50
mL), dried over MgSO4, filtered and concentrated in vacuo to give 2.82 g of yellow foam.
Purification by flash chromatography using a 150 g disposable silica gel column and 0→80%
EtOAc in CH2Cl2, gave the pure material (1.24 g) as an off-white solid. Rf = 0.23 in 50%
1
EtOAc:CH2Cl2. H NMR (400 MHz, CDCl3):  7.51(S, 1H), 5.54(s, 1H), 1.57(m, 2H),
1.44(br s, 9H), 1.18(m, 2H). 13C NMR (101 MHz, CDCl3):  174.0, 160.5, 155.8, 155.3,
148.5(t, J = 26.77 Hz), 135.69(t, J = 24.31 Hz), 120.1(t, J = 23.57 Hz), 119.0(t, J = 23.57 Hz),
80.0, 50.8, 49.7(m), 35.6, 28.2, 19.1, 17.8(m).
(S)-2-Amino-N-(1-pyridin-2H4-2-yl-cyclopropyl)-propionamide-2H4, [2H8]-(7): To
a
solution of [2H8]-(12) (0.86 g, 2.75 mmol) in CH2Cl2 (10 mL) was added a solution of 4M
HCl in 1,4-dioxane (7 mL) dropwise. The resulting mixture was stirred at room temperature
for 4 h. TLC (10% MeOH/CH2Cl2) showed no starting material and the presence of polar
material at the baseline. The mixture was concentrated in vacuo to give an off-white solid
1
(0.8 g crude material). LCMS, one single peak (MH+ = 214.49). H NMR (400 MHz,
DMSO-d6):  7.3(s, 1H), 5.54(brs, 4H), 1.61(m, 2H), 1.20(m, 2H).
(R)-3-(4-Cyanobenzyl)-1-(3,5-dichloro-4-fluorophenyl)-3-methyl-2-oxo-N-((S)-1-oxo-1-
((1-(pyridin-2H4-2-yl)cyclopropyl)amino)propan-2-yl)-2,3-dihydro-1H-imidazo[1,2-
a]imidazole-5-carboxamide ethyl-2H4, [2H8]-(2): To a solution of acid (6) (1.26 g, 2.75
mmol) and amine [2H8]-(7) (0.8 g, 2.75 mmol) in anhydrous DMF (25 mL) was added HATU
(1.25 g, 3.3 mmol) in one portion. To this solution, Hünig's base (2.4 mL, 13.6 mmol) was
added dropwise and the reaction was stirred at room temperature for 14 h. The reaction was
o
concentrated at 40 C to remove most of the DMF, then diluted with CHCl3 (200 mL) and
washed with water (200 mL). The organic phase was dried over MgSO4, filtered and
concentrated in vacuo to give 3 g of viscous oil which contained some DMF. Purification by
This article is protected by copyright. All rights reserved.

flash chromatography using a 150 g disposable silica gel column and 0→10% MeOH/CH2Cl2
gave 1.32 g (72%) of product. HPLC, tR = 10.47 min (99%) co-eluted with unlabeled
1
standard,. TLC: 10% MeOH/CH2Cl2: Rf = 0.5. HNMR (400 MHz, CDCl3) : 7.70(d, J =
5.74 Hz, 2H). 7.50(s, 1H), 7.38(d, J= 8.06 Hz, 2H), 7.30(s, 1H), 7.15(s, 1H), 6.98(d, J= 8.11
Hz, 2H), 4.13(d, J= 13.48 Hz, 1H), 3.34(d, J = 13.48 Hz, 1H), 1.94(brs, 3H), 1.65(m, 2H),
1.31(m, 2H). 13CNMR (101 MHz, CDCl3) : 174.59, 173.49, 160.14, 159.15, 154.05, 151.55,
148.77(t, J = 26.77 Hz), 147.94, 139.77, 135.87 (t, J = 24.31 Hz), 132.25, 131.92, 130.08,
128.90, 128.86, 123.66, 122.95, 122.77, 122.01, 120.55(t, J = 23.57 Hz), 118.67(t, J = 23.57
Hz), 118.28, 111.57, 69.18, 53.47, 50.69, 48.92(m), 42.45, 36.54, 22.42, 18.96, 16.96(m).
2
HRMS, calculated for C32H19 H8O3N7Cl2F: 654.20331, found 654.20325.
Synthesis of [13C5,15N]-(3):
(tert-Butoxycarbonyl)-L-threonine-13C4,15N, [13C4,15N]-(13): L-Threonine-13C4,15N (1 g,
8.1 mmol) was mixed with methanol (12 mL) and a solution of sodium bicarbonate (1.04 g,
12.37 mmol) in water (12 mL) at room temperature. Di-tert-butyl-dicarbonate (2.57 g, 11.8
mmol) was added in one portion and the mixture was stirred for 30 h. LCMS showed all the
starting material was consumed. The mixture was concentrated in vacuo to remove most of
the solvents and the residue was diluted with water (50 mL) and extracted with ether (50 ml
×2). The aqueous layer was made acidic, pH = 3 by adding 1.0 N aqueous HCl, and extracted
with Me-THF (50 ml ×2). The combined extracts were dried over Na2SO4, filtered and
concentrated in vacuo to give 2.1 g of colorless oil in quantitative yield. LCMS, Rt = 1.34 min,
1
MH+ = 225.0 (100%), EI-, M- = 223.1 (100%). H NMR (400 MHz, CDCl3) : 6.1(brs 2H),
13
5.73(m, 1H), 4.15-4.52(m, 2H), 1.42(brs, 9H), 1.16(m, 3H). C NMR (101 MHz, CDCl3) :
174.42(d), 156.5, 80.2, 67.92(m), 60.2(m), 58.64(m), 28.2, 19.47(d).
tert-Butyl
((2S,3R)-1-((1-(5-(1,3-dimethyl-1H-pyrazol-4-yl)pyrimidin-2-
yl)cyclopropyl)amino)-3-hydroxy-1-oxobutan-13C4-2-yl)carbamate-15N, [13C4,15N]-(15):
To a mixture of (14) (2.4 g, 7.94 mmol) and [13C4,15N]-(13) (1.77 g, 7.9 mmol) in anhydrous
CH2Cl2 (80 mL) was added Hünig's base (8.3 mL, 47.65 mmol) slowly to give a colorless
solution. HOBT (1.2 g, 8.88 mmol) and EDC (1.7 g, 8.87 mmol) were added and the solution
was stirred at room temperature for 14 h. Most of the solvent was removed in vacuo and the
residue was diluted with EtOAc (200 mL), and washed with 1.0 N aq. HCl (100 mL). The
aqueous yellow solution was removed and the organic was washed with a saturated aqueous
NaHCO3 (100 mL), brine (100 mL), dried over Na2SO4, and concentrated. Purification on
Combi-Flash using 120 g silica gel column and up to 20% MeOH/CH2Cl2 gave 2.2 g of an
This article is protected by copyright. All rights reserved.

off-white solid in 80% yield. LCMS: tR = 0.69 min, MH+ = 436.0 (100%), M- = 434.1 (100%).
1H NMR (400 MHz, CDCl3) : 8.56(s, 2H), 7.45(s, 1H), 7.06(brs, 1H), 5.2-5.8(m, 1H), 3.8-
4.6(m, 1H), 3.49(m, 1H), 2.34(m, 2H), 1.87(m, 1H), 1.4-1.7(m, 2H), 1.47(brs, 9H), 1.13(m,
3H). 13C NMR (101 MHz, CDCl3) : 173.2(d), 166.7, 154.5, 145.9, 129.3, 128.9, 125.3,
124.5, 120.3, 113.5, 109.3, 80.1, 67.2(m), 62.8(m), 60.2(m), 58.6(m), 38.8, 36.7, 28.3(m),
18.4(d).
(2S,3R)-1-((1-(5-(1,3-dimethyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclopropyl)amino)-3-
hydroxy-1-oxobutan-13C4-2-amine-15N, [13C4,15N]-(8): To a solution of [13C4,15N]-(15) (1.3
g, 3.0 mmol) in anhydrous CH2Cl2 (30 mL), was added a solution of 4N HCl in 1,4-dioxane
(3.0 mL, 12 mmol) slowly at room temperature. A precipitate was formed instantly. The
mixture was stirred at room temperature for 5 h, and then concentrated in vacuo. The white
solid residue was treated with anhydrous ether (30 mL) and filtered. The solid was washed
with anhydrous ether (10 mL ×2), collected, transferred to a round bottom flask, and dried at
o
35 C for 4 h and then under reduced pressure overnight to give 1.2 g of a white solid in
quantitative yield. LCMS, tR = 0.19 min, MH+ = 336.0 (100%). 1H NMR (400 MHz, DMSO-
d6) : 9.4(d, J = 4 Hz, 1H), 8.74(brs, 2H), 8.30(br, 2H), 8.12(br, 2H), 3.44(3, 3H), 2.51(m,
13
2H), 2.3(s, 3H), 1.25-160(m, 4H), 1.2(m, 3H). CNMR (101 MHz, DMSO-d6) : 168.58(d,
J = 50 Hz), 154.12, 144.59, 130.39, 124.35, 112.73, 66.3(t, J = 37 Hz), 58.34(ddd, J = 37, 36,
52 Hz), 38.36, 36.10, 19.40, 12.80. (R)-5-(4-Cyano-benzyl)-7-(3,5-dichloro-4-fluoro-
phenyl)-5-methyl-6-oxo-6,7-dihydro-5H-imidazo[1,2-a]imidazole-3-carboxylic-13C acid,
[13C]-(6): Anhydrous DMF (2 mL) was added to a vial containing carbon-13 sodium formate
(352 mg, 5.05 mmol) followed by Hünig's base (0.7 mL, 4.0 mmol) and acetic anhydride (0.4
mL, 4.0 mmol) and stirred at rt. Stirring was continued for 30 min at room temperature, the
mixture become thick Gas bubbles were observed. LiCl (263 mg, 6.2 mmol) was dissolved
in anhydrous DMF (4 mL) by sonication in a separate vial to give a clear solution. To this
vial, were added the iodide (5) (1.1 g, 2.0 mmol) and Pd(OAc)2 (90 mg, 0.4 mmol). The
o
resulting solution was added to the sodium formate vial and the mixture was heated to 80 C
and stirred for 14 h. LCMS showed all the starting material was consumed and the presence
of one major product co-elutes with the unlabeled acid standard (6). The dark mixture was
diluted with EtOAc (50 mL) and treated with 2.0 N of aqueous HCl (40 mL). The organic
phase was removed and washed with brine (40 mL ×2), dried over MgSO4, filtered, and
concentrated in vacuo to give 1.2 g of foam. Purification by silica gel chromatography using
40 g disposable column and 10% to 30% EtOAc:CH2Cl2 gave 0.75 g of pure product as an
This article is protected by copyright. All rights reserved.

off-white solid in 81% yield. LCMS, Rt = 1.76 min, MH+ = 459.48 (100%), 460.48(80%).
1HNMR (400 MHz, CDCl3) : 10.2(s, 1H), 8.1(s, 1H), 7.64(d, J = 5.8 Hz, 2H), 7.44(d, J =
8.21 Hz, 2H), 7.01(d, J = 8.21 Hz, 2H), 3.48(d, J = 13.64 Hz, 1H), 3.38(d, J = 13.64 Hz, 1H),
1.98(s, 3H).
(R)-3-(4-Cyanobenzyl)-1-(3,5-dichloro-4-fluorophenyl)-N-((2S,3R)-1-((1-(5-(1,3-
dimethyl-1H-pyrazol-4-yl)pyrimidin-2-yl)cyclopropyl)amino)-3-hydroxy-1-oxobutan-
13C4-2-yl)-3-methyl-2-oxo-2,3-dihydro-1H-imidazo[1,2-a]imidazole-5-carboxamide-
13C,15N, [13C5,15N]-(3): The acid [13C]-(6) (461 mg, 1.0 mmol) was dissolved in anhydrous
THF (10 mL). Two drops of DMF were added and the solution was cooled in an ice-bath.
Oxalyl chloride (152 mg, 1.2 mmol) was added dropwise and the resulting solution was
warmed gradually to room temperature and stirred for 14 h. HPLC of a sample quenched with
morpholine showed only the morpholine derivative. The reaction was concentrated in vacuo
to foam. The amine [13C4,15N]-(8) (372 mg, 1.0 mmol) was suspended in anhydrous THF (10
mL). Hünig's base (260 mg, 2.0 mmol) was added dropwise at room temperature to give a
colorless solution with some white precipitate. The mixture was stirred for 1 h. The above
acylchloride was dissolved in THF (6 mL) and added dropwise to the reaction flask
containing the amine [13C4,15N]-(8). The flask was washed with THF (4 mL) and added to the
o
reaction. After stirring for 1 h at 0 C, the ice-bath was removed and the mixture was stirred
for 14 h at ambient temperature. The reaction was quenched with water (30 mL) and MTBE
(50 mL) was added. The aqueous (bottom) phase was removed and the organic was washed
with 2 % aqueous NaHCO3 (50 ml ×2), dried over Na2SO4, filtered and concentrated in vacuo
to give 0.9 g of a yellow solid. Purification using 40 g disposable silica gel column and up to
5% MeOH/CH2Cl2 gave 669 mg of pure material as an off-white solid in 86% yield. LCMS:
tR = 1.06 min, MH+ = 775.6 (30%), 776.8 (100%), 779.8 (30%). EI-, M- = 774.1 (30%),
775.0(100%), 777.9(30%). HPLC: Halo-C18 (2.1 x 50 mm, 2.7 m), 20% to 100% B in 9
min, A = H2O (0.1% TFA), B= MeCN, tR = 5.83 min, 99.25% at 230 nm. HRMS: MH+ m/z
777.2263 observed, calculated m/z 777.2258; HPLC area % purity 99.1%. 1H NMR (400
MHz, CDCl3) : 8.59(s, 2H), 7.71(d, J = 4 Hz, 2H), 7.60(d, J = 28 Hz, 2H), 7.55(s, 1H),
7.48(s, 1H), 7.43(m, 1H), 7.41(d, J = 8 Hz, 2H), 7.15(s, 1H), 7.02(d, J = 8.0 Hz, 2H), 5.55(q,
J = 7 Hz, 0.5 H), 5.30(m, 0.5 H), 4.95(m, 0.5 H), 4.79(m, 0.5 H), 4.60(m, 0.5 H), 4.42(m, 0.5
H), 4.16(d, J = 12Hz, 1H), 3.90(s, 3H), 3.58(q, J = 5 Hz, 1H), 3.37(d, J = 12Hz, 1H), 2.35(s,
3H), 1.94(s, 3H), 1.78(m, 2H), 1.56(m, 2H), 1.47(d, J = 4 Hz, 1H), 1.44(t, J = 6 Hz, 2H,
13
1.28(m, 4H). C NMR (101 MHz, CDCl3) : 172.01(d, J = 51 Hz), 159.25(d, J = 50 Hz),
This article is protected by copyright. All rights reserved.

154.79, 148.19, 145.97, 139.90, 131.91, 130.88, 129.06, 128.86, 124.85, 123.08, 122.81,
118.24, 113.54, 111.63, 69.30, 68.14(t, J = 38 Hz), 58.54(ddd, J = 11.06, 35.22, 41.30 Hz),
52.87, 42.31, 38.94, 36.94, 22.67, 18.20(d, J = 40.25 Hz), 14.12, 12.99.
Conclusion:
A two-step radiosynthesis route was used to prepare two highly potent LFA-1 antagonists
labeled with carbon-14 with specific activities higher than 56 mCi/mmol and radiochemical
purities of 99%. The synthesis included a palladium catalyzed [14C]carbonylation of an iodide
derivative using carbon-14 sodium formate and amide bond formation between the carbon-14
labeled carboxylic acid and the corresponding chiral primary amine derivatives (7) and (8).
Deuterium labeled (2) was prepared in four steps using commercially available deuterium
labeled
2-cyanopyridine
which
was
subjected
Kulinkovich-Szymoniak
aminocyclopropanation followed by amide bond formation with deuterium labeled BOC-L-
alanine, then removal of the BOC-protecting group and coupling to acid (6). The availability
of L-threonine-13C4,15N allowed the preparation of the amine [13C4,15N]-(8) in only three steps
with 80% overall yield. Coupling this amine to [13C]-(6) gave [13C5,15N]-(3) in very good
yield. In conclusion, we report our approach to the preparation of these chiral compounds
with rather complicated structures in fewer steps and provide valid tools for drug metabolism,
pharmacokinetics, and other studies.
This article is protected by copyright. All rights reserved.

References
[1] (a) Latli B, Byrne D, Nummy L, Krishnamurthy D, Senanayake C.H. Synthesis of
potent lymphocyte function-associated antigen-1 inhibitors labeled with carbon-14
and deuterium, Part 1. J. Label. Compds. Radiopharm. 2011; 54:763-768.
(b) Latli B, Hrapchak M, Xu Y, Qiu F, Krishnamurthy D, Senanayake C H Synthesis
of highly potent lymphocyte function-associated antigen-1 inhibitors labeled with
stable isotopes and carbon-14, Part 2. J. Label. Compds. Radiopharm. 2011; 54:799-
808.
[2] Lemieux R M, Brunette, S R, Horan, J C et al. Derivatives of 6,7-dihydro-5H-
imidazo[1,2-a]imidazole-3-carboxylic acid amides. US 20120252817 A1
[3] Frutos R P, Tampone T, Mulder J A, et al. A new and practical boronic acid catalyzed
intramolecular cyclodehydration of ureas for the synthesis of LFA-1 anatagonists.
Tetrahedron Lett. 2011; 52:2465-2467.
[4] Cacchi S, Fabrizi G, Goggiamani A. Palladium-catalyzed hydrocarbonylation of aryl
and vinyl halides or triflates by acetic anhydride and formate anions. Org. Lett. 2003;
5:4269-4272.
[5] Berger P, Bessmernykh A, Caille A-C, Mignonac S. Palladium-catalyzed
hydrocarbonylation of aryl and vinyl bromides by mixed acetic and formic anhydride.
Synthesis 2006; 18:3106-3110.
[6] Elmore C S, Dean D C, Melillo D G. A convenient method for [14C]carbonylation
reactions. J. Label. Compds. Radiopharm. 2000; 43:1135-1144.
[7] Elmore C S. The synthesis and the use of [14C]carbon monoxide in Pd-catalyzed
carbonylation reactions. J. Label. Compds. Radiopharm. 2011; 54:59-64.
[8] Nielsen D U, Neumann K T, Lindhardt A T, Skrydstrup T. Recent developments in
carbonylation chemistry using [13C]CO, [11C]CO, and [14C]CO. J. Label. Compd.
Radiopharm. 2018;1–39. Special Issue, https://doi.or g/10.1002/jlcr.3645
[9] Li W, Gao J, Lornz J C, et al. Process development and pilot-plant synthesis of (S)-
tert-butyl 1-oxo-1-(1-(pyridin-2-yl)cyclopropylamino)propane-2-ylcarbamate: Studies
on the scale-up of Kulinkovich-Szymoniak cyclopropanation. Org. Process Res. Dev.
2012; 16:836-839.
[10] Bertus P, Szymoniak J. Ti(II)-Mediated conversion of -heterosubstituted (O, N, S)
nitriles to functionalized cyclopropylamines. Effect of chelation on the
cyclopropanation step. J. Org. Chem. 2002; 67:3965-3968.
This article is protected by copyright. All rights reserved.

[11] Solorzano C, Antonietti F, Duranti A, et al. Synthesis and structure-activity
relationships of N-(2-oxo-3-oxetanyl)amides as N-acylethanolamine-hydrolyzing acid
amidase inhibitors. J. Med. Chem. 2010; 53:5770-5781.
[12] The synthesis of this intermediate will be published elsewhere.
This article is protected by copyright. All rights reserved.

Figure 1: Structural modifications made on the new LFA-1 inhibitors
This article is protected by copyright. All rights reserved.

Figure 2: HRMS of unlabeled (2) and [2H8]-(2)
This article is protected by copyright. All rights reserved.

Scheme 1: Synthesis of [14C]-(2) and [14C]-(3)
This article is protected by copyright. All rights reserved.

Scheme 2: Synthesis of [2H8]-(2)
This article is protected by copyright. All rights reserved.

Scheme 3: Synthesis of [13C5,15N]-(3)
This article is protected by copyright. All rights reserved.

TOC:
Two highly potent LFA-1 inhibitors (2) and (3) were efficiently prepared labeled with
carbon-14 using a palladium-catalyzed carboxylation of an iodo-precurssor (5) and sodium
formate-14C followed by amide bond formation with chiral amines (7) and (8). These two
drug candidates were also labeled with stable isotopes by first preparing the chiral amines
[2H8]-(7) and [13C4,15N]-(8) and then coupling them to (6) and [13C]-(6).
This article is protected by copyright. All rights reserved.